The mitogen associated proteins (MAP) kinase cascade has been characterized in rat fibroblasts transformed by a ts isolate of Moloney murine sarcoma virus (Mo-MuSV) (ts110), as well as in a newly-isolated, flat (non-transformed) v-mos revertant (F-1). Using a ts-transformed cell line, it has been shown that MAP kinase activity was expressed at the permissive temperature (34 degreesC), but not at the non-permissive temperature (39 degreesC). Appearance of both ERK-2 (MAP kinase) and MKK-1 (MAP kinase kinase-1) activity following a 39 degrees to 34 degrees shift is coincident with the appearance of mos-specific kinase activity. It was found that reexpression of v-mos was sufficient to fully activate ERK-2 and MKK-1 in serum-starved cells at the permissive temperature. In contrast, despite the presence of intact and fully active v-mos, revertant F-1 cells exhibited low ERK-2 and MKK-1 activity. Moreover, F-1 cells were found to express elevated levels of protein phosphatase in comparison to normal and parental transformed cells, and treatment of F-1 cells with okadaic acid, a phosphatase inhibitor, leads to a significantly increased level of MAP kinase activity in cell lysates. Spontaneous revertants of F-1 cells, which had required the transformed phenotype, also expressed constitutively elevated levels of activated MAP kinase. The results support the hypothesis that mos acts through the MAP kinase cascade (MKK-1 and ERK-2) to induce cell transformation. Blocking v-mos activation of that cascade (possibly increased levels of phosphatase-inactivated components of the pathway) prevents transformation by mos. A NIH3T3 cell variant, SFDH, selected for serum-free growth, had been shown to undergo a cyclic spheroid/monolayer transition, spontaneously form spheroids after reaching confluence, and the spheroids spreading and reforming a monolayer culture upon replating in fresh dishes and media. Spheroid formation and maturation correlates with increased expression of met, hepatocyte growth factor, and the epithelial marker cytokeratin.